T-cell responses to ancestral SARS-CoV-2 and Omicron variant among unvaccinated pregnant and postpartum women living with and without HIV in South Africa

SARS-CoV-2 cell-mediated immunity remains understudied during pregnancy in unvaccinated Black African women living with HIV (WLWH) from low- and middle-income countries. We investigated SARS-CoV-2-specific T-cell responses 1 month following infection in 24 HIV-uninfected women and 15 WLWH at any stage during pregnancy or postpartum. The full-length spike (FLS) glycoprotein and nucleocapsid (N) protein of wild-type (WT) SARS-CoV-2, as well as mutated spike protein regions found in the Omicron variant (B.1.1.529) were targeted by flow cytometry. WT-specific CD4+ and CD8+ T cells elicited similar FLS- and N-specific responses in HIV-uninfected women and WLWH. SARS-CoV-2-specific T-lymphocytes were predominantly TNF-α monofunctional in pregnant and postpartum women living with and without HIV, with fever cells producing either IFN-γ or IL-2. Furthermore, T-cell responses were unaffected by Omicron-specific spike mutations as similar responses between Omicron and the ancestral virus were detected for CD4+ and CD8+ T cells. Our results collectively demonstrate comparable T-cell responses between WLWH on antiretroviral therapy and HIV-uninfected pregnant and postpartum women who were naïve to Covid-19 vaccination. Additionally, we show that T cells from women infected with the ancestral virus, Beta variant (B.1.351), or Delta variant (B.1.617.2) can cross-recognize Omicron, suggesting an overall preservation of T-cell immunity.


Fig. S7. Maternal polyfunctionality of CD8+ T-cells to the FLS and N proteins of WT SARS-CoV-2 in women living with HIV.
(a) Comparisons of the polyfunctional profiles of CD8 + T cells to the FLS and N proteins of WT SARS-CoV-2 in WLWH, stratified by maternal status.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.(b) Each response pattern is colour-coded, and data summarized in pie charts.The Mann-Whitney U-test was used for comparing unpaired CD8 + T-cell responses of positive responders between pregnant, postpartum, and non-pregnant women.Only significant response differences are indicated (p<0.05).(c) Correlations between FLS-and N-specific CD4 + T-cell responses according to IL-2, IFN-γ, and TNF-α production.Spearman's rank correlation coefficient (two-sided) was used to determine the P and r (correlation coefficient) values.Linear regression lines are indicated in red, together with 95% confidence interval lines.Denotations: FLS, fulllength spike glycoprotein; IFN-γ, interferon gamma; IL-2, interleukin 2; N, nucleocapsid protein; TNF-α, tumor necrosis factor alpha; WLWH, women living with HIV; WT, wild-type (ancestral) SARS-CoV-2.Comparisons of individual cytokine median fluorescence intensities (MFIs) of (a) CD4 + and (b) CD8 + T cells in response to the FLS-and N-proteins of WT SARS-CoV-2, stratified by HIV status.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.The Mann-Whitney U-test was used for comparing unpaired MFIs between women living with and without HIV.The Wilcoxon singed-rank test and Friedman test were used for comparing MFIs between two and three paired groups, respectively.Denotations: FLS, full-length spike glycoprotein; IFN-γ, interferon gamma; IL-2, interleukin 2; MFI, median fluorescence intensity; N, nucleocapsid protein; TNFα, tumor necrosis factor alpha; WLWH, women living with HIV; WT, wild-type (ancestral) SARS-CoV-2.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.The Mann-Whitney U-test was used for comparing unpaired MFIs between women living with and without HIV.The Wilcoxon singed-rank test and Friedman test were used for comparing MFIs between two and three paired groups, respectively.Denotations: FLS, full-length spike glycoprotein; IFN-γ, interferon gamma; IL-2, interleukin 2; MFI, median fluorescence intensity; N, nucleocapsid protein; TNF-α, tumor necrosis factor alpha; WLWH, women living with HIV; WT, wildtype (ancestral) SARS-CoV-2.

Fig. S2 .
Fig. S2.Overall median comparisons of SARS-CoV-2-specific CD4 + and CD8 + T-cell responses in women living with and without HIV.Direct median comparisons between CD4 + and CD8 + T-cell polyfunctionality profiles in response to the (a) FLS and (b) N proteins on WT SARS-CoV-2 among women living with and without HIV.Each point represents the median T-cell response of positive responders for each group of cytokines produced.CD4 + T-cell responses are indicated in green and blue.CD8+ T-cell responses are indicated in red and orange.The Wilcoxon signed-rank test was used for comparing paired CD4 + and CD8 + T-cell responses.Significant P-values are indicated in bold (p<0.05).Denotations: FLS, full-length spike glycoprotein; IFN-γ, interferon gamma; Il-2, interleukin 2; N, nucleocapsid protein; TNF-α, tumor necrosis factor alpha; WLWH, women living with HIV; WT, wild-type (ancestral) SARS-CoV-2.

Fig. S3 .
Fig. S3.Maternal T-cell responses to the FLS and N proteins of WT SARS-CoV-2.The proportion of T-cells producing IL-2, IFN-γ, or TNF-α in response to the FLS and N proteins of WT SARS-CoV-2, stratified by maternal (pregnant: n=17; postpartum: n=16; not pregnant: n=5) and HIV status (HIV-uninfected: n=23; WLWH: n=15).SARS-CoV-2-specific CD4 + T-cell responses to (a) the FLS and (b) N proteins.SARS-CoV-2-specific CD8 + T-cell responses to (c) the FLS and (d) N proteins.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.Statistical analysis was performed using the Mann-Whitney U-test for T-cell response comparisons between unpaired (HIV-uninfected vs. WLWH) responders.Significant P-values are indicated in bold (p<0.05).Denotations: FLS, full-length spike glycoprotein; HIV-, HIV-uninfected women N, nucleocapsid protein; WLWH, women living with HIV; WT, wild-type (ancestral) SARS-CoV-2.

Fig. S4 .
Fig. S4.Maternal polyfunctionality of CD4 + T-cells to the FLS and N proteins of WT SARS-CoV-2 in HIV-uninfected women.(a) Comparisons of the polyfunctional profiles of CD4 + T cells to the FLS and N proteins of WT SARS-CoV-2 in HIV-uninfected women, stratified by maternal status.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.(b) Each response pattern is colour-coded, and data are summarized in pie charts.The Mann-Whitney U-test was used for comparing unpaired CD4 + T-cell responses of positive responders between pregnant and postpartum

Fig. S5 .
Fig. S5.Maternal polyfunctionality of CD4 + T-cells to the FLS and N proteins of WT SARS-CoV-2 in women living with HIV.(a) Comparisons of the polyfunctional profiles of CD4 + T cells to the FLS and N proteins of WT SARS-CoV-2 in WLWH, stratified by maternal status.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.(b) Each response pattern is colour-coded, and data are summarized in pie charts.The Mann-Whitney U-test was used for comparing unpaired CD4 + T-cell responses of positive responders between pregnant, postpartum, and non-pregnant women.Only significant response differences are indicated (p<0.05).(c) Correlations between FLSand N-specific CD4 + T-cell responses according to IL-2, IFN-γ, and TNF-α production.Spearman's rank correlation coefficient (two-sided) was used to determine the P and r (correlation coefficient) values.Linear regression lines are indicated in red, together with 95% confidence interval lines.Denotations: FLS, full-length spike glycoprotein; IFN-γ, interferon gamma; IL-2, interleukin 2; N, nucleocapsid protein; TNF-α, tumor necrosis factor alpha; WLWH, women living with HIV; WT, wild-type (ancestral) SARS-CoV-2.

Fig. S6 .
Fig. S6.Maternal polyfunctionality of CD8 + T-cells to the FLS and N proteins of WT SARS-CoV-2 in HIV-uninfected women.(a) Comparisons of the polyfunctional profiles of CD8 + T cells to the FLS and N proteins of WT SARS-CoV-2 in HIV-uninfected women, stratified by maternal status.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.(b) Each response pattern is colour-coded, and data summarized in pie charts.The Mann-Whitney U-test was used for comparing unpaired CD8 + T-cell responses of positive responders between pregnant and postpartum women.Only significant response differences are indicated (p<0.05).(c) Correlations between FLS-and N-specific CD8 + T-cell responses according to IL-2, IFN-γ, and TNF-α production.Spearman's rank correlation coefficient (two-sided) was used to determine the P and r (correlation coefficient) values.Linear regression lines are indicated in red, together with 95% confidence interval lines.Denotations: FLS, fulllength spike glycoprotein; HIV-, HIV-uninfected women; IFN-γ, interferon gamma; IL-2, interleukin 2; N, nucleocapsid protein; TNF-α, tumor necrosis factor alpha; WT, wild-type (ancestral) SARS-CoV-2.

Fig. S8 ,
Fig. S8, Cytokine median fluorescence intensities of CD4 + and CD8 + T-cells to the FLS and N proteins of WT SARS-CoV-2 in women living with and without HIV.Comparisons of individual cytokine median fluorescence intensities (MFIs) of (a) CD4 + and (b) CD8 + T cells in response to the FLS-and N-proteins of WT SARS-CoV-2, stratified by HIV status.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.The Mann-Whitney U-test was used for comparing unpaired MFIs between women living with and without HIV.The Wilcoxon singed-rank test and Friedman test were used for comparing MFIs between two and three paired groups, respectively.Denotations: FLS, full-length spike glycoprotein; IFN-γ, interferon gamma; IL-2, interleukin 2; MFI, median fluorescence intensity; N, nucleocapsid protein; TNFα, tumor necrosis factor alpha; WLWH, women living with HIV; WT, wild-type (ancestral) SARS-CoV-2.

Fig. S9 ,
Fig. S9, Cytokine median fluorescence intensities of CD4 + and CD8 + T-cells to WT SARS-CoV-2 and the Omicron variant in women living with and without HIV.Comparisons of individual cytokine median fluorescence intensities (MFIs) of (a) CD4 + and (b) CD8 + T cells in response to spike proteins of WT SARS-CoV-2 and the Omicron variant, stratified by HIV status.Each participant is represented by an individual point, and medians in each group are indicated by horizontal lines.The Mann-Whitney U-test was used for comparing unpaired MFIs between women living with and without HIV.The Wilcoxon singed-rank test and Friedman test were used for comparing MFIs between two and three paired groups, respectively.Denotations: FLS, full-length spike glycoprotein; IFN-γ, interferon gamma; IL-2, interleukin 2; MFI, median fluorescence intensity; N, nucleocapsid protein; TNF-α, tumor necrosis factor alpha; WLWH, women living with HIV; WT, wildtype (ancestral) SARS-CoV-2.